The IkB kinase complex (IKK) plays a central role in the immune and inflammatory responses by controlling the activation of NF?kB transcription factors. Dysregulation of the IKK/NF-kB pathway is associated with numerous diseases such as diabetes, cancer, inflammatory and autoimmune diseases; therefore IKK has become an attractive drug target for the treatment of these common human diseases. Here we propose to address two fundamental questions on IKK: 1) What is the mechanism of IKK activation? 2) What are the underlying mechanisms of IKK substrate specificity and catalysis? Previous studies have demonstrated that NEMO is indispensible for IKK? activation, and recent structural studies have indicated that the IKK? dimers have distinct inactive and active structural conformations. Furthermore, our preliminary data have suggested that NEMO forms a large molecular assembly with IKK?. We hypothesize that IKK? undergoes at least two transitional stages during its activation, a pre-activation complex and a post-activation (activated) complex. In the pre- activation complex, the activation segments of neighboring kinase domains are able to contact each other for trans-autophosphorylation. While in the post-activation complex, large conformational changes occur to allow the kinase domains to swing away from each other, making room for substrate binding and catalysis. We propose to determine the structures of the pre- and post-activation NEMO- IKK? holo-complexes to reveal NEMO-mediated IKK? trans-activation. In addition, we have invented a novel approach to utilize the IKK?-specific viral inhibitor protein B14 to specifically target IKK? activation but not kinase activity, thereby enhancing our understanding of the mechanism of IKK activation. Unveiling the mechanism of NEMO-mediated IKK activation, as well the mechanism of B14-mediated IKK? inhibition, will lead to novel strategies to target IKK activation or specific inhibition of IKK? activation while ot affecting its basal cellular activity and IKK?. These novel strategies will help enhance drug potency, specificity, and reduce side effects of disease treatment. In addition, we propose to unveil the structural basis of the docking interaction between IKK and IkB and investigate the influence of docking interaction on IKK substrate specificity and catalytic mechanism. These studies will not only aid the optimization of current IKK inhibitors but also lead to novel strateges of targeting IKK-substrate docking interaction.